Biomedical research is being revolutionized by breakthrough new capabilities in the separation, identification, and structural characterization of the genome's expressed proteins. The "bottom up" approach of proteolysis of the protein mixture and MS/MS of its peptides is now widely used for protein identification. However, we have championed the "top down" approach and our powerful addition, electron capture dissociation (ECD). These not only provide orders-of-magnitude higher reliability in identifying a protein from those DMA- predicted, but are far more specific and sensitive in pinpointing posttranslational modifications and sequence errors. Of our 30 papers published in the last 4-year grant period, 17 show a wide variety of such applications to biosynthesis, enzyme mechanism, protein folding, and kinetics problems, with such research on-going. A major future research effort will be in expanding the top down applicability from 74 kDa to far larger proteins using segmented nozzle-skimmer dissociation;we have already achieved 287 interresidue cleavages for a 144 kDa protein and 62 for a 229 kDa. A very promising recent discovery is that "folding inhibition adducts'to the electrospray solution can increase cleavages by 50%;understanding the mechanism should give further improvements. A wild idea could do ECD during electrospray. We shall also try to extend the far higher top down identification reliability to such far larger proteins, defining its capabilities as a possible replacement for bottom up identifications. The current progress was made possible by our research showing that electrosprayed proteins form "ball of spaghetti" noncovalently bound conformers that must be denatured before backbone dissociation. We have found new methods for such studies that we will continue to exploit. Infrared photodissociation spectroscopy of 4 proteins and mutants show unexpectedly large red shifts of N-H and O-H absorptions, indicating that H- bonding is far stronger in gaseous ion conformers than in native solution conformers. Segmented nozzle skimmer dissociation separately cleaves noncovalent and covalent bonds, while inlet capillary heating finds intractable transient conformers. ECD data define conformer structures for protein ions, while native ECD details the unfolding of a native conformer during electrospray.